Anti myc mouse monoclonal antibody

Xenopus laevis oocytes were injected with cRNA of noninactivating (Δ6–46) Shaker K_V channels (Hoshi et al., 1990 (link)) that contained a c-myc epitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) inserted at the C terminus (after Val638) and incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, and 5 HEPES, pH 7.6) at 17°C. 24 h after injection, oocytes were exposed to 10 mg/ml PNGase F diluted 1:10 in ND96. The enzyme activity was monitored by removing 10 oocytes at different times, supplemented with 100 mM glycine and lysed in 200 µl buffer H (1% Triton X-100, 100 mM NaCl, and 20 mM Tris-HCl, pH 7.4). Lysates were rocked at room temperature for 15 min and then centrifuged at 13,000 rpm for 3 min. The pellet was discarded, and the supernatant was analyzed by Western blot by using anti–Myc mouse monoclonal antibody at a dilution of 1:5,000 (Clontech) and detected with a secondary, goat anti–mouse antibody conjugated to horseradish peroxidase at a dilution of 1:10,000 (Pierce). Membranes were developed by SuperSignal WestFemto (Thermo Fisher Scientific) and visualized by chemiluminescence by using a FluorChem E Imager (Cell Biosciences).